Method and system for the production of cells and cell products and applications thereof

ABSTRACT

A cell culture system for the production of cells and cell derived products includes a reusable instrumentation base device incorporating hardware to support cell culture growth. A disposable cultureware module including a cell growth chamber is removably attachable to the instrumentation base device. The base device includes microprocessor control and a pump for circulating cell culture medium through the cell growth chamber. The cultureware module is removably attached to the instrumentation base device. Cells are introduced into the cell growth chamber and a source of medium is fluidly attached to the cultureware module. Operating parameters are programmed into the microprocessor control. The pump is operated to circulate the medium through the cell growth chamber to grow cells or cell products therein. The grown cells or cell products are harvested from the cell growth chamber and the cultureware module is then disposed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of:

U.S. patent application Ser. No. 12/274,993, filed Nov. 20, 2008, whichis incorporated herein by reference in its entirety and which is acontinuation in part of:

International Application No. PCT/US2007/012042, filed May 21, 2007,which claims the benefit under 35 USC §119 of U.S. Application No.60/802,376, filed May 22, 2006, both of which are incorporated herein byreference in their entirety;

International Application No. PCT/US2007/012051, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006, both of which are incorporated herein by reference in theirentirety;

International Application No. PCT/US2007/012052, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006, both of which are incorporated herein by reference in theirentirety;

International Application No. PCT/US2007/012053, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006, both of which are incorporated herein by reference in theirentirety; and

International Application No. PCT/US2007/012054, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006; both of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and system that creates aself-contained culture environment, and more particularly to a cellculture system incorporating a disposable cultureware module and areusable compact instrumentation base device that is capable ofexpanding cells including primary cells and cell lines as well aspatient-specific cells or cells lines in an automated, contaminant-freemanner.

2. Description of the Related Art

The anticipated growth of personalized medicine will require newparadigms for the manufacture of therapies tailored to the needs ofindividual patients. The greatest challenge is expected to come in thearea of cell based therapies, especially when such therapies areautologous in nature. In such cases each cell or cell based product willneed to be manufactured from scratch for each patient. Manual methodsfor mammalian cell culture, by their nature, are prone to technicianerror or inconsistency leading to differences between supposed identicalcultures. This becomes especially evident as more and more autologouscells are expanded for personalized therapies. Patient-specific cells,or proteins, are subject to variation, especially when scaled beyondlevels that can be managed efficiently with manual methods.

In addition to being labor intensive, the stringent requirements forsegregation of each patient's materials from that of every other patientwill mean that manufacturing facilities will be large and complex,containing a multitude of isolation suites each with its own equipment(incubators, tissue culture hoods, centrifuges) that can be used foronly one patient at a time. Because each patient's therapy is a new andunique product, patient specific manufacturing will also be laborintensive, requiring not just direct manufacturing personnel but alsodisproportionately increased manpower for quality assurance and qualitycontrol functions.

Moreover, conventional approaches and tools for manufacturing cells orcell based products typically involve numerous manual manipulations thatare subject to variations even when conducted by skilled technicians.When used at the scale needed to manufacture hundreds or thousands ofdifferent cells, cell lines and patient specific cell based therapies,the variability, error or contamination rate may become unacceptable forcommercial processes.

Small quantities of secreted product are produced in a number ofdifferent ways. I-flasks, roller bottles, stirred bottles or cell bagsare manual methods using incubators or warm-rooms to provideenvironments for cell growth and production. These methods are verylabor intensive, subject to mistakes and difficult for large scaleproduction.

Another method, ascites production, uses a host animal (usually a mouse)where the peritoneum is injected with the cells that express the productand are parasitically grown and maintained. The animals are sacrificedand the peritoneal fluid with the product is collected. This method isalso very labor intensive, difficult for large scale production andobjectionable because of the use of animals. Another method is toinoculate and grow the cells in a small stirred tank or bioreactor orbag-type chamber. The tank provides the environmental and metabolicneeds and the cell secretions are allowed to accumulate. This method iscostly in terms of facility support in order to do a large number ofunique cells and produces product at low concentration.

Another method is to use a bioreactor (hollow fiber, ceramic matrix,fluidizer bed, etc) in lieu of the stirred tank. This can bringfacilities costs down and increases product concentration. BiovestInternational of Coon Rapids, Minn., has or had instruments using thesetechnologies—hollow fiber, ceramic matrix, fluidized bed and stirredtanks.

Cell culturing devices or cultureware for culturing cells in vitro areknown. As disclosed in U.S. Pat. No. 4,804,628, the entirety of which ishereby incorporated by reference, a hollow fiber culture device includesa plurality of hollow fiber membranes. Medium containing oxygen,nutrients, and other chemical stimuli is transported through the lumenof the hollow fiber membranes or capillaries and diffuses through thewalls thereof into an extracapillary (EC) space between the membranesand the shell of the cartridge containing the hollow fibers. The cellsthat are to be maintained collect in the extracapillary space. Metabolicwastes are removed from the bioreactor. The cells or cell products canbe harvested from the device.

Known EC reservoirs have typically been rigid. They are a pressurevessel and therefore require a sealed compartment with tubing portsadding to costs. A gas, typically air, is introduced through a sterilebarrier, generally a membrane filter, to control pressure in the vessel.Fluid level control has been limited to ultrasonic, conductive oroptical trip points, or by a load cell measuring the weight of thefluid. Reservoirs are expensive and difficult to manufacture. There islimited EC fluid level measurement accuracy—ultrasonic, conductive oroptical monitoring of fluid levels are commonly fouled by cell debris inthe reservoir. Alternatively, load cells are not a rugged design forreliable fluid level sensing.

Another problem with the prior art systems is the inability to controllactate and sense pH in the system. One prior art method takes samplesof the culture medium and analyzes it using an off-line analyzer. Theoperator adjusts the perfusion medium rate based on values obtained tomaintain the lactate concentration at the level desired. The operatormust attempt to predict future lactate levels when adjusting media feedrates. This is labor intensive, presents potential breech of sterility,and the level of lactate control accuracy is dependent on operatorskill.

Another method is to connect an automated sampler/analyzer toperiodically withdraw sample of the culture media, analyze it andprovide feedback for a media feed controller. This method requiresadditional equipment and increases the risk of sterility breech.

Yet another method is to use an invasive lactate sensor to directly readthe lactate level and provide feedback for a media feed controller. Inline lactate sensors need to be sterilizable, biocompatible, typicallyhave low reliability and need periodic maintenance.

These methodologies rely on costly, labor intensive off-line samplingand analysis or additional equipment to interface with the instrument orrequire the addition of a lactate probe and electronics to the culture.

Disposable cultureware generally cannot be autoclaved, so a pH sensor ishistorically sterilized separately and then added to the cultureware.However, adding the probe risks compromising the sterility of thecultureware. Probe addition is performed in a sterile environment(laminar flow hood) and increases the manpower needed.

The previous methodologies that utilize off-line sampling are subject tocontamination problems and depend on the skill of the operator inpredicting future lactate levels and influence of media dilution rate.Sampling equipment need interfacing to the culture fluidic circuit, aninterface for the feedback signal and periodic calibration of the probesused for sampling. The lactate probe requires interface with the fluidcircuit, a method for sterilization or a sterile barrier, interfaceelectronics to convert the probe signal to a useful feedback and amethod to calibrate in the fluid circuit.

Preparing the system to start the cell culture is also very laborintensive. The cultureware must be assembled and sterilized or probesmust be prepared, sterilized and aseptically inserted into thepre-sterilized portion of the cultureware. The cultureware assembly isthen loaded onto the instrument. A series of manual operations areneeded to check the integrity of the assembly, introduce fluid into thecultureware flow path, flush the toxic residuals (e.g. surfactants) fromthe cultureware, start the cultureware in a pre-inoculation mode,introduce factors into the flow path getting it ready for the cells,inoculating the cells into the bioreactor and starting the run (growthof the cell mass and eventual harvest of product).

Two methods are generally used for sterilization. One method places anelectrode in a holder, steam sterilizes the assembly (probe) and thenaseptically inserts the probe into the pre-sterilized cultureware. Thesecond method involves placing a non-sterile probe into a holder andthen using steam to sterilize the electrode in place, referred to assteam in place. Both methods are labor intensive, prone to failure andthe procedures need to be validated.

Other methods exist which are less common. Cold sterilants can be usedto sterilize the holder and electrode before aseptic insertion. Apermeable membrane can be used to isolate the non-sterile probe from thesterile fluid being sensed. A holder with the membrane is placed in thefluid path, either before sterilization or after if the holder andmembrane is sterilized separately, and then the sensor is placed againstor close to the membrane and the fluid on both sides of the membrane isassumed to be equilibrated.

Glass electrodes have not been included with the cultureware in the pastbecause it was unknown if the probes could survive EtO sterilization andbeing stored dry. Filled glass electrodes are normally stored hydratedin a liquid buffer.

Each unique cell or cell line must be cultured, cell products harvestedand purified separately. In order to do a large number of unique cellsor cell lines, a considerable number of instruments would be needed. Ifapplication of the cells or products for therapeutic purposes iscontemplated strict segregation of each cell production process would berequired. Consequently, compactness of the design and the amount ofancillary support resources needed will become an important facilitiesissue. Moreover the systems currently available are general purpose innature and require considerable time from trained operators to setup,load, flush, inoculate, run, harvest and unload. Each step usuallyrequires manual documentation.

Moreover, production tracking mandates generation of a batch record foreach cell culture run. Historically this is done with a paper-basedsystem and relies on the operator inputting the information. This islabor intensive and subject to errors.

Current purification techniques also involve cleaning and reuse ofcomponents. This requires Standard Operational Procedures (SOPS) to bewritten and the cleaning and reuse process to be validated. This is atime intensive activity.

Accordingly, there is a need for a system and method whereby cellsand/or cell products can be cultured in a fully automated, rapid andsterile manner.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a modular andintegrated system for the production and expansion of cells or celllines. The system consists of a reusable control module housing with allof the mechanical and electronic components and disposable cell growthmodules that attach to the control module. This system minimizes theneed for skilled technicians and more importantly, prevents thepossibility of cross-contamination in a multi-use facility. As anenclosed system, the safety provided by complete segregation facilitatesdirect applicability to therapies or diagnoses that require autologouscell culture. This self-contained, automated cell culture device allowsfor simultaneously culture of numerous cell cultures within a compactfacility, without the need for individual, segregated cell culturesuites. The system of the present invention provides a compact sealedcontainment system that will enable the cost effective manufacture ofcells, cell lines, patient specific cells and cell products on anindustrial scale.

Another aspect of the present invention is to provide a method andsystem that incorporates disposable cultureware, which eliminates theneed for cleaning and reuse.

Yet another aspect of the present invention is a system that has thestand-alone integration of a large system in a bench top device (pumps,controls, incubator, refrigerator, cultureware, etc).

Still another aspect of the present invention is a system thatincorporates a barcode reader and data gathering software that, whenused with an information management system (such as a manufacturingexecution system or MIMS), allows for automating generation of the batchrecord.

Another aspect of the present invention is to provide an EC cycling unitthat costs less than rigid reservoirs. Moreover, due to the sealed ECcircuit design, without vented reservoir, the chance of cellcontamination is minimized.

Still another aspect of the present invention is to provide a systemthat controls lactate concentration in a perfusion cell culture systemusing measurement of CO₂ and pH.

Yet another aspect of the present invention is to eliminate preparation,autoclaving, and insertion of pH electrodes aseptically in thecultureware which requires a significant amount of time and may breachthe sterile barrier of the cultureware set.

The system of the present invention incorporates features that greatlyreduce the operator's time needed to support the operations (e.g.integrated pump cassette, pre-sterilized cultureware with pH sensors,quick-load cultureware) and designed automated procedures andapparatuses which allow the system to sequence through the operations(e.g. automated fluid clamps, control software).

The system is capable of integrating the cell culture product productionand purification process. The design of the cultureware and instrumentsimplifies and reduces labor needed to produce product. This reducessources of error in the process.

The present invention provides an automated cell culture system andmethod which creates a self-contained culture environment. The apparatusincorporates perfusion culture with sealed, pre-sterilized disposablecultureware, such as hollow fiber or other bioreactors, programmableprocess control, automated fluid valving, pH feedback control, lacticacid feedback control, temperature control, nutrient delivery control,waste removal, gas exchange mechanism, reservoirs, tubing, pumps andharvest vessels. Accordingly, the present cell culture apparatus(referred to as AutovaxID Cell Culture Module™) is capable of expandingcells in a highly controlled, contaminant-free manner. Cells to whichthis approach are applicable include transformed or non-transformed celllines, primary cells including somatic cells such as lymphocytes orother immune cells, chondrocytes, myocytes or myoblasts, epithelialcells and patient specific cells, primary or otherwise. Included alsoare cells or cell lines that have been genetically modified, such asboth adult and embryonic stem cells. Specifically, the automated cellculture apparatus allows for production and harvest of cells orcell-secreted protein in a manner that minimizes the need for operatorintervention and minimizes the need for segregated clean rooms for thegrowth and manipulation of the cells. Further, the apparatus provides aculture environment that is completely self-contained and disposable.This eliminates the need for individual clean rooms typically requiredin a regulated, multi-use facility. Control of fluid dynamics within thebioreactor allows for growth conditions to be adjusted, e.g. changinggrowth factor concentrations, to facilitate application of uniqueculture protocols or expansion of unique cells or cell lines. As aresult, there is less variation and less labor required for consistent,reproducible production of cells for applications to expansion ofautologous cells and their use in personalized medicine applications.

According to these and other aspects of the present invention, there isprovided a cell culture system for the production of cells and cellderived products including a reusable instrumentation base deviceincorporating hardware to support cell culture growth. A disposablecultureware module including a cell growth chamber is removablyattachable to the instrumentation base device.

According to these and other aspects of the present invention, there isalso provided a method for the production of cells and cell products ina highly controlled, contaminant-free environment comprising the stepsof providing a disposable cultureware module including a cell growthchamber, and a reusable instrumentation base device incorporatinghardware to support cell culture growth. The base device includesmicroprocessor control and a pump for circulating media through the cellgrowth chamber. The cultureware module is removably attached to theinstrumentation base device. Cells are introduced into the cell growthchamber. A source of media is fluidly attached to the culturewaremodule. Operating parameters are programmed into the microprocessorcontrol. The pump is operated to circulate the media through the cellgrowth chamber to grow cells or cell products therein. The grown cellsor cell products are harvested from the cell growth chamber. Thecultureware module is then disposed.

These and other features, aspects, and advantages of the presentinvention will become more apparent from the following detaileddescription of the preferred embodiment relative to the accompanieddrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the system for producing cells and/orcell derived products according to the present invention.

FIG. 2 is another perspective view of the system of the presentinvention.

FIG. 3 is a perspective view of the instrumentation device of thepresent invention.

FIG. 4 is a rear and partial side view of the device with covers removedof FIG. 3.

FIG. 5 is a rear view of the device with covers removed of FIG. 3.

FIG. 6 is an enlarged view of the heating system of the device of FIG.3.

FIG. 7 is a perspective view of the variable output pump of the systemof the present invention.

FIG. 8 is an exploded view of the pump of FIG. 7.

FIG. 9 is a perspective view of the pump cassette of the system of thepresent invention

FIG. 10 illustrates the installation method of the cell culture moduleand the device of the present invention.

FIG. 11 is a perspective view of the gas blending and fluid cyclingcontrol of the module of the present invention.

FIG. 12 is a front view of the fluid cycling control of FIG. 11.

FIG. 13 is a perspective view of a rotary selection valve drive of thepresent invention.

FIGS. 14A and 14B are exploded views of the valve rotor of FIG. 13. andthe body used with it. FIG. 14C is a rear view of the valve body.

FIGS. 15A-15C are perspective views of a tubing slide clamp of thepresent invention

FIG. 16 is a perspective view of the factor and harvest bags of thepresent invention.

FIG. 17 is a perspective view of the disposable culture medium module ofthe present invention.

FIG. 18 is an interior view of the module of FIG. 17.

FIG. 19 is a perspective interior view of the back of the module of FIG.17.

FIG. 20 is a perspective view of the extra-capillary cycling unit of thepresent invention.

FIG. 21A is a flow diagram of the cycling unit of FIG. 20.

FIG. 21B is an exploded view of the cycling unit.

FIG. 22 is a flow diagram of the lactate control system of the presentinvention.

FIG. 23 is a flow diagram of the system of the present invention.

FIG. 24 is another perspective view of the module of the presentinvention.

FIG. 25 is a cross-sectional view of a flexible hollow fiber bioreactoraccording to the present invention.

FIGS. 26-31 are views of the touch screen associated with the automaticcontrol of the system of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 1, the present invention provides a fully integratedsystem 10 for producing cells and cell derived products in a closed,self-sufficient environment. More specifically, the system allows forcell expansion and harvest of cells and their products with minimal needfor technician interaction. As will be described further herein, thedevice incorporates cell culture technology, for example, hollow fiberor similar bioreactor perfusion technology, with all tubing componentsother than the media feed, harvest tubing and tubes threaded through thepump cassette, encased in a single-use, disposable incubator 12.Following bioreactor inoculation with cells, the system followspre-programmed processes to deliver media, maintain pH, maintain lactatelevels, control temperature and harvest cells or cell-secreted protein.Standard or unique cell culture growth parameters can be programmedprior to bioreactor inoculation, such that, various cell types can beexpanded and such that cells or cell products can be harvested in anefficient, reproducible manner with minimal chance of human error.

The system is based on cell growth chamber technology. For example,bioreactors that have a plurality of semi-permeable hollow fibers orother type of semi-permeable membrane or substrate potted in a housingto create a space inside the fiber or one side of the membrane (referredto as intracapillary or IC space) separate from that outside the fibersor on the other side of the membrane (referred to as extracapillary orEC space). Fluid distribution between the IC and EC space occurs throughthe fiber pores which can range in size from 10 MW(Kd) to 0.2 μm. Cellsare placed on one side of the fiber or membrane, usually in the ECspace, in a complete cell culture medium, which is usually the samemedium used to expand cells prior to bioreactor inoculation (serumcontaining, serum-free, or protein-free medium). Cells are usuallyplaced in the EC space when secreted protein is the desired product. Insome instances, when cells are the desired product, it may be beneficialto place cells in the IC space.

Medium is perfused through a bioreactor 20 by circulating through the ICspace at a fast rate. The medium can be a liquid containing awell-defined mixture of salts, amino acids, and vitamins that oftencontains one or more protein growth factors. This serves to delivernutrients to the cell space and conversely, removes or prevents a toxicbuild-up of metabolic waste. During this circulation, medium is passedthrough an oxygenator or gas exchanger cartridge 24 which serves toprovide pH control and oxygen for the cells and conversely, removecarbon dioxide from the culture. When the bioreactor 20 contains asmaller number of cells, just after inoculation, the oxygenator or gasexchange cartridge is used to provide CO₂ and subsequently control pH ofthe culture environment. As cell number increases, the oxygenator isused to remove CO₂ which serves to enhance acid neutralization andcontrol the pH of the culture. Other bioreactor configurations, inaddition to hollow fibers, that are designed and optimized for thegrowth and production of cells and production of cell-derived productsare also included.

The system 10 provides significant efficiencies and cost reductionthrough its disposable component and enclosed operation. As such, celllines are contained in a closed system and continuously cultured withoutthe need for specialized, segregated clean rooms. This fully integratedapparatus eliminates the need for cleaning and sterilizationvalidations, as well as the need for hard plumbing associated withconventional cell culture facilities.

Referring again to FIG. 1, the system consists of two individual parts:an instrumentation base device 14 that is reusable and an enclosedcultureware module 12 that is used for a single production run and isdisposable. Numerous modules 12 can be used on a single device 14. Theinstrument provides the hardware to support cell culture growth andproduction in a compact package. As shown in FIG. 2, and as will bedescribed in further detail herein, an easy-load multiple channelperistaltic pump drive 16 located in base device 14 and a pump cassette70 move fresh basal media into the cultureware, removes spent media,adds growth factors or other supplements and removes product harvest. Anintegrated cool storage area 18 maintains the factor and harvest at alow temperature (approximately 4° C.). An integrated heating mechanism22 (FIG. 6) maintains the cell environment to promote growth andproduction. Gas exchange cartridge 24 (FIG. 5), in conjunction with acultureware pH sensor 26 controls the pH of the cell culture medium. Twoautomated tube valving drives 90 (FIG. 3) are used to control thecultureware flow path configuration to accomplish the fluidic switchingfunctions needed to initiate and do a successful run. Valves 90 andsensors 32 (FIGS. 3, 5, 13) in the instrument control the fluid cyclingin the cultureware module 12. A drive pump 34 (FIGS. 3, 5) for fluidcirculation is provided. An attached barcode reader, not shown,facilitates operator and lot tracing. A communication port ties theinstrument to a data information management system (such as a MES). Aflat panel display 36 (FIG. 1) with touch screen is available for userinteraction.

The one-time use cultureware module 12 is provided pre-sterilized. It isdesigned for quick loading onto the instrument (“quick-load”), as willbe described further herein. The loading of the cultureware body makesconnections to the instrument. Pump cassette 70 (FIG. 2), which isphysically attached to the tubing, allows the user to quickly load thepump segments. This design and layout minimizes loading errors. Thecultureware enclosure 12 provides an area that is heated to maintaincell fluid temperature. A fluid cycling unit 40 (FIGS. 1, 18) maintainsfluid volumes and cycling and is included in the cultureware. Sensorsfor fluid circulation rate, pH and a thermal well for the instrument'stemperature sensor are provided. The blended gas from the instrument isrouted to gas exchange cartridge 24 that provides oxygen and adds orremoves carbon dioxide to the circulated fluid to support cellmetabolism. A magnetically coupled pump drive 34 (FIGS. 11-12)circulates fluid thru the bioreactor 20 and gas exchange cartridge 24.The bioreactor 20 that provides the cell space and media componentexchange is also in the cultureware. Disposable containers for harvestcollection are provided. Prior to the beginning of the culture theoperator attaches a media source, factor bag and spent media containerto the cultureware before running. At the conclusion of the run theharvest containers are removed or drained, media and spent mediacontainer is disconnected, pump cassette is unloaded, harvest bagdisconnected, cultureware body is unloaded and the used cultureware isplaced in a biohazard container for disposal.

Cell expansion and subsequent process tracking mandates generation of abatch record for each culture. Historically this is done with apaper-based system that relies on operator input of the information.This is labor intensive and subject to errors. The fully integrateddevice incorporates a barcode reader and data gathering software which,when used with the information management system (MES), allows forautomatic generation of the batch record.

The system of the present invention has application in a regulated cellculture environment. It is anticipated that autologous whole celltherapies or patient-specific proteins (vaccines) therapies, would bytheir nature, require the simultaneous culture of numerous cell lines ina single facility. In addition to the segregation created through thisclosed culture approach, the apparatus is designed to support a standardinformation management system (such as a LIMS or MES) protocol. Thiscapability contributes to the creation of thorough batch records andverification of culture conditions to ensure standardization, trackingand safety of each product. This capability facilitates themulti-product concept that is pivotal to facilities involved withautologous or patient-specific products.

Referring to FIG. 1, disposable cell culture module 12 is removablyattachable to device 14. The module requires multiple mechanical andelectrical interfaces to the control instrumentation of device 14.Module 12 has interface features integrated into the module that matewith instrument interface features in the device to allow for a singlemotion installation (FIG. 10). As modules 12 are to be disposed of afteruse, it should be appreciated that numerous modules can be used inconjunction with a single base device 14.

As shown in FIG. 3, the interface features of device 14 includecirculation pump drive 34, actuator valves 90 and cycling sensor 32. Inaddition, a temperature probe 44 and a flow sensor 46 interface with thecomponents of module 12. Device 14 also includes an electricalconnection 48 for pH probe 26 disposed within module 12.

Gas ports 52 communicate with gas exchanger 24. One port 52 communicateswith the input to exchanger 24 and the other port 52 communicates withthe output of the exchanger. Gas ports 54 control pressure to thecycling fixture 40. One port 54 communicates with the IC chamber and theother port 54 communicates with the EC space. As viewed from the front,the left port 52 is the exchanger output and the right port 52 is theexchanger input. The top port 54 is the IC reservoir pressurizationport, and the lower port 54 is the EC reservoir pressurization port.

As described above, module 12 is heated to maintain cell fluidtemperature. Heating mechanism 22 (FIG. 6) maintains the cellenvironment to promote growth and production. The cell culture,disposable modules 12 requiring elevated temperatures are warmed byfully encapsulating the module and attaching the module to thecontrolling instrument 14, such that air ports are aligned and warmedair is forced into the module from the instrument at one location andallowed to escaped at another. Instrument device 14 has a heated airoutlet 58 and a return heated air inlet 56.

When disposable module 12 is installed onto the controlling instrumentdevice 14, the air inlet 88 (FIG. 19) of the disposable module alignswith the air outlet 58 of the controlling instrument. Heating mechanism22 forces warmed air through outlet 58 and into the warmed air inlet 88and into disposable module 12. The warmed air elevates the temperatureof the components inside of the module. The exhaust air exits throughair outlet 86 and into air inlet 56 of instrument device 14 where it iscirculated through recirculated.

During installation, module 12 is aligned with the connections of thedevice 14 and the module is placed into the operating position as shownin FIG. 10. All mating interface features are functional. Referring toFIG. 19, when installed, certain features of the module 12, formed in aback panel 148 of the module, interface with device 14. Module airoutlet 86 aligns with device air inlet 56 and module air inlet 88 alignswith device air outlet 58 to circulate heated air through module 12 asdescribed herein. Gas connectors 152 and 154 engage device gas ports 52and 54, respectively, to allow gas to enter and exit module 12. Valvebodies 156 receive actuator valves 90. Hub 158 receives pH probe 26interface and aligns with electrical connector 48. Module 12 isconnected to circulation pump drive 34 via module pump connection 164.Cycling unit 40 also communicates with cycling sensor 32 when the moduleis installed. The flow sensor 46 of device 12 mates with flow sensorconnection 166. The temperature sensor 44 of device 14 mates with a noninvasive receptacle in module 12 that is in contact with the IC media toprovide control feed back to the control mechanism to regulate thethermal output of heater 22. The above mating connections facilitate theone-motion installation of the module 12 on the device.

Referring to FIGS. 7-9, the present invention incorporates amulti-position, cassette loading, and peristaltic pump 16 (FIG. 2) withdiscrete, variable output control for each channel. A plurality ofchannels 60 (FIG. 3) are located in device 14. Although four channelsare shown, it should be appreciated that pump 16 could have more or lesschannels.

As shown in FIG. 8, the pump has individual, variable control of theoutput of each channel. Pump rotors 62A-62D have a common fixed axialshaft 64 with individual servo drive. The occlusion rotors 66A to 66Dare mounted to the pump rotors 62A-62D, which in turn are mounted on thesingle shaft 64 with internal bearings that allow for independentfunctional control by a respective reacting servo drive 68A-D. Thesingle shaft minimizes tolerance accumulations typically caused bymisalignment of individual rotors and shafts mating with a multi-channelcassette. Feedback sensors are included to verify rotation of the pumprotors.

Typical multi-channel peristaltic pump applications operate using arotating drive shaft that is common to all rotors. This causes allrotors to turn at the same revolution per minute (RPM), yielding thesame fluid output. Different inside diameter tubing may be used to givea fixed ratio delta output from one rotor to another. To obtain avariable output of the peristaltic pump segments, individual pump headsand drives are used. This requires individual tubing cassettes that mustbe loaded individually and does not allow for close center to centerdistance between pump heads.

As shown in FIG. 9, a multi-channel cassette 70 is featured withpre-loaded peristaltic tubing 72 to reduce loading errors and to reduceinstallation time. The mechanism includes a cam operated cassetteinsertion feature 74) that interfaces with 67 on pump 16. As shown inFIG. 8, a knob 65 is rotated to move cam feature 74 into position to aidinitial tubing occlusion during loading.

The cassette configuration is structured to hold multiple peristaltictubing segments. A gripping feature 76 on the top and the bottomprevents the tubing from creeping during operation. The design allowsfor all tubing segments to be loaded into the pump drive mechanism atthe same time. A latching feature 74 is also included to provide abearing surface for the cam-operated latch 67 to react upon.

Referring back to FIGS. 1 and 2, cassette 70 is pre-loaded withperistaltic tubing (FIG. 24) and positioned in groove 80 on module 12.After module 12 is positioned on device 14, cassette 70 is removed andinserted into interface or plate 82. Each cassette section 71 (FIG. 9)supporting the tubing is inserted into a respective channel 60 (FIG. 3)of the interface 82. This configuration reduces tubing segment loadingerrors with pre-loaded multi-position cassettes, and reducesinstallation time.

Referring to FIGS. 11 and 12, valves and sensors 32 in the instrumentcontrol the fluid cycling in the cultureware module 12. Two opticalsensors detect the low or high position of the cycling position sensorflag 140 (FIG. 20). This information is used by a predictive algorithmto control the pressures applied to the IC chamber and EC pressure bagto effect cycling.

Sterilizable, disposable, actuator driven, rotary selection valves 90are shown in detail in FIGS. 13-14C. Valve 90 comprises a valve housing93 and valve cam 94. The elastomer tubing (not shown) is insertablethrough openings 98 in valve body 92 and is occluded by a rotating cam94 that compresses the tubing against the valve body. This isaccomplished by using a controlled, incremental, servo drive 96(actuator and position feed back loop) to move cylindrical cam thatreacts against immobile valve body 92 that holds the tubing in aconstrained state. The cam design allows for a high area of the cam 104to occlude the tubing and a low area of the cam 106 not to occlude,resulting in a closed and open condition respectively. Cam rotationalpositioning features may also be added to move cam 94 to predeterminedpositions. Configurations can be structured to accommodate multipletubing segments in one device. The two piece design allows for fluidcontact portion of the valve to be molded into the backpanel 148 (FIG.19) as a hub 156 and to be sterilized (EtO, chemical or radiation) withthe rest of the fluid circuit and eliminates the need to be addedseparately.

The design of this clamp is meant to be used in an automated cellculture application where a disposable cultureware module interfaceswith an electro-mechanical instrument. The combined unit is to beautomated, which required various tubing lines of the disposable to beoccluded/open to provide automated process control. The selector valveis used to automatically open and close tubing lines to direct fluid orgas flow during process control. Minimizing operator set-up is also arequirement. The disposable cultureware must be inserted into theinstrument in an operating position with no special operator proceduresrequired for loading the tubing into the clamps. Existing technologiesdid not meet these requirements, because the manual clamps were notautomated, and solenoid valves required a special operator loadingprocedure.

In the cell culture system of the present invention, the fluid path mustbe free of unwanted organisms (sterilized). Commercially availableselector valves are not gas sterilizable. Sealing surfaces of theselected position may be unexposed to the gas sterilant and thosesurfaces may be “non-sterile” when the valve is repositioned. Valve 90provides automated actuation of the cam, compactness, multiple lines,maintains valve position even with loss of actuator power, thedisposable valve body is less costly than an equivalent switching valve,and can be incorporated into the back panel of 12. Offset occluded/opencam positioning of two tubing lines can insure a make-before-breakswitching of fluids. No power is required to maintain any operatingposition, and tubing segments used in the valve body can be sterilized.

It should be appreciated that a solenoid driven pinch mechanism, can beused in place of the actuator valve. This application may utilize apiston plunger actuated by an electrical coil to provide linear motionto pinch the tubing. A manual pinch clamp could also be used. Theclamping position is manually activated by a mechanical bearing surfacecompressing the tubing and then held in position by a detent feature.This clamp type requires manual deactivation. A membrane over the seriesof ports could also be used. The membrane is actuated against the portto seal it. Multiple ports are configured for use as a selectormechanism.

In another embodiment shown in FIGS. 15A-15C, an actuator driven tubingslide clamp 110 with multiple positions and multiple tubing can be usedas an alternative to valves 90. Elastomer tubing is occluded by slidingthe tubing into a narrow slot 112 that compresses the tubing wallagainst itself. This is accomplished by using a servo drive (actuatorand position feed back loop) to move a plate 110 with slot 112 in it andreacting against another plate or slide body 114 that holds the tubingin an immobile state. The moveable plate is designed with varying widthslots to allow for position/positions to be inactive. This allows fornormally open 116 or 118 and normally closed 117 positions.Configurations can be structured to accommodate multiple tubing segmentsin one clamp.

In operation, slide 110 is positioned into slide body 114. Tubing isinserted through tubing ports 108 and slide 110 at position 116 whereboth tubes are not occluded. A remote servo (not shown) engages intoserver drive slot 102 and moves the slide to position 117 where one tubeis occluded and one tube is not occluded. The remote Servo than movesthe slide to position 118 where the occluded tube from the previous stepis not occluded, and the tube from the not occluded tube from theprevious step is now occluded. When moving the slide from position 117to position 118, both tubes are occluded to insure that one tube isoccluded before the other tube is opened. It should be appreciated thatthe number of tubes and configuration of the slide can be modified tomeet customized applications.

The clamp is meant to be used in an automated cell culture applicationwhere a disposable cultureware module interfaces with anelectro-mechanical instrument. The combined unit is to be automated,which required various tubing lines of the disposable to beoccluded/open to provide automated process control. During processcontrol the clamps are open/closed to simulate the function of anexpensive, “disposable” switching valve. Minimizing operator set-up isalso a requirement. The disposable cultureware must be inserted into theinstrument in an operating position with no special operator proceduresrequired for loading the tubing into the clamps. It provides automatedactuation of slide clamp, compactness, multiple lines, maintains clampposition even with loss of actuator power, less costly than anequivalent switching valve. Offset occluded/open position of two tubinglines can insure a make-before-break switching of fluids. No powerrequired to maintain any operating position.

As described above, integrated cool storage area 18 maintains growthfactors and harvested cells or cell products at a low temperature(approximately 4° C.). Referring to FIGS. 2 and 16, a rack 120 isremovably positionable within cool storage area 18. Rack 120 is designedto support a plurality of bags 122, 124. The bags are used to containthe smaller quantities of product or growth factors. It should beappreciated that other solutions can be disposed with the bags. Forexample, high molecular weight growth factor can be located with bag122. This factor is connected via tubing 128 to the bioreactor or cellgrowth chamber 20 and the flow controlled by pump 16. Harvested cells orcell products can be stored in bag 124. A cell filter 126 is provided toprovide additional filtration. A filter bypass line is included iffiltering of the harvest is not desired as in the case of cellcollection. After the process is complete the cells can be removed fromthe cell culture chamber via the tubing and stored in bag 124 until use.

As shown in FIGS. 17-19, disposable cultureware module 12 includes fluidcycling unit 40 to maintain fluid volumes and cycling in the cell growthchamber. Referring to FIGS. 18-21, the present invention utilizesextra-capillary (EC) cycling in cell culture growth chamber 20 (FIG. 17)utilizing a non-rigid, EC reservoir 130 and mechanical or a secondflexible reservoir 132 to cause elevated EC pressure. Reservoirs 130,132 are separated by a sensor plate 134. Reservoirs 130, 132 arerestricted in the maximum amount of expansion by a rigid mechanicalhousing 136. EC cycling is achieved by utilizing a non-rigid reservoirto retain the varying fluid volume associated with an EC circuit.Flexible reservoir 130 is fluidly connected to the bioreactor or hollowfiber device 20. Second flexible reservoir 132 is pressurized to applyforce against the flexible reservoir 130 to provide an elevated ECpressure to cause an ultra-filtrative condition and force fluid into anintra-capillary (IC) circuit 138. A mechanical feed back positionindicator 140 is physically connected to sensor plate 134 and moves withthe physical expansion and contraction of the first flexible reservoir.The position of indicator 140 is sensed by the position sensors 32 andis used to control the force that is applied by second flexiblereservoir 132. It should be appreciated that an alternate mechanicalforce apparatus may be used instead of a second flexible reservoir tocause pressure changes.

During operation the pressure is increased in the IC circuit 138 bypressurizing an IC reservoir 137. This pressure causes anultra-filtrative condition that forces fluid transmembrane across thesemi-permeable matrix of the bioreactor 20. The fluid is then forcedthrough the connect tubing, through a flow control valve 133 and intothe EC reservoir 130. Externally controlled pressure in the pressurereservoir 132 is allowed to vent. The expanding EC reservoir 130 forcesthe sensor plate 134 toward the pressure reservoir 132 and compressesit. Sensor plate 134 moves external position flag 140 and this is sensedwhen EC reservoir 130 has filled enough to expand to the EC upper level.The external position sensor 32 senses this position and the pressure inthe IC reservoir 137, is decreased and the pressure in the pressurereservoir 132 is increased. This causes an ultra-filtrative conditionand forces fluid out of the EC reservoir through a control valve 135,transmembrane across the matrix of the bioreactor 20 and into the ICcircuit 138. The sensor plate 134 moves the external position flag 140and the sensor 32 senses when the EC reservoir 130 has contracted to theEC low level.

The EC cycling unit of the present invention offers fluid dynamics tocause fluid flow in the EC space thus minimizing nutrient and metabolicwaste gradients that may be detrimental to the cells. It provides fluidlevel control without the use of ultrasonics or load cells that is notaffected by cell debris. The flexible reservoirs are considerably lessexpensive and are suited for disposable applications. The sealed ECreservoir with cycling also limits contamination and isolates the cells.

The present invention also includes an indirect lactate control methodfor perfusion culture using CO₂ and pH sensing. The method predicts opensystem, perfusion culture, lactate levels in the circulatory medium bymonitoring the pH and off-gas CO₂ level. This is accomplished bycalculating the initial bicarbonate level of the media then utilizingthe liquid pH and gas level of CO₂ to calculate current lactateconcentration. This is used to control media dilution rate of the cellculture. The resulting calculated lactate value is used to set theperfusion rate of media dilution to maintain a pre-determined lactatelevel. Thus, an invasive sensing system or multiple off-line sampling isnot required.

A physical relationship exists between bicarbonate buffer, dCO2, and pH.

pH=pK+log([HCO₃ ⁻ ]/dCO_(2]))  Equation (1):

where:

-   -   pH=the pH of the solution    -   pK=the acid ionization constant for bicarbonate    -   HCO₃ ⁻=the current bicarbonate concentration (mM)    -   dCO₂=the concentration of dissolved CO₂

Lactic acid production by the cells appears to be the dominant drivingforce for pH changes in cell culture media. Based on this observation,each mole of lactic acid produced results in consumption of one mole ofbicarbonate as described by the following equation:

[HCO₃ ⁻]=[HCO₃ ⁻]₀−[Lactate]  Equation (2)

where:

-   -   [HCO₃ ⁻]₀=the initial bicarbonate concentration in the medium        (mM)    -   Lactate=the lactate concentration (mM)

Equation (3) provides a simple relationship—Henry's Law, thatequilibrium dCO₂ is proportional to the gas phase concentration of CO2.

dCO₂ =a(%CO₂)  Equation (3):

where:

-   -   a=CO₂ solubility conversion (mM/%)    -   % CO₂=concentration of CO₂) in the gas phase that is in        equilibrium with dCO₂ (%).

Equation (4) is derived by substituting Equation 2 in Equation 1 asfollows:

pH=pK+log {([HCO₃ ⁻]₀−[Lactate])/[dCO₂]}  Equation (4)

Equation 5 is derived by combining Equations 3 and 4:

pH=pK+log {([HCO₃ ⁻]₀−[Lactate])/[a(%CO₂)]}  Equation (5)

The operating equation, Equation (6) is derived by solving for Lactatein Equation (5):

Lactate=[HCO₃ ⁻]₀−(a)*(%CO₂)*10^((pH-pK))  Equation (6):

The values of pK and (a) were found to be 6.38 and 0.39, respectively.

Upon taking a lactate and pH reading, the value of (a) is calculated.The initial bicarbonate concentration is calculated as the calibrationconstant. The advantage is that the bicarbonate concentration does nothave to be known when using the present calibration method.

The application is shown in FIG. 22. In a bioreactor perfusion loop, thegrowth media is pumped from an IC reservoir 137 via pump drive 34, 164,circulated to the gas exchange cartridge (GEX) 24, pH sensor 26,bioreactor 20, and then back to reservoir 137.137. Blended gases arepassed through the membrane gas exchange cartridge that oxygenates themedia and regulates CO₂. Per Henry's Law, the CO₂ levels in the gasphase or air side of the GEX 24 is in equilibrium with the liquid phaseof the media. The discharge end of the GEX is monitored with a CO₂sensor 142 that resides in the device 14 and the lactate is calculatedper Equation (6). When the media lactate level is known, the instrumentuses automatic, media dilution, control to maintain the predeterminedset point.

The present invention utilizes existing signals and with the addition ofa non-invasive gas CO₂ sensor incorporates lactate control to controlmedia feed rate for cell growth and production. Utilizing the inventionreduces materials and labor associated with recurring off-line testing.Utilizing the invention allows for continual adjustment of the dilutionrate that would otherwise be inefficient and costly if step increaseswere used as in previous technologies.

Utilizing the present invention increases the predictability of cellculture metabolics. Allows a perfusion cell culture system to have anincreased level of automation. The lactate and media dilution rate canbe used to determine the state of cell growth and production.

The present invention also utilizes a novel approach for pH sensing in acell culture system. Referring back to FIGS. 2, 18 and 19, pH probe 26and a holder are built into cell culture disposable 12, thus the user isnot required to add the probe to the cultureware. Probe 26 is intendedto be a one-time use device that is disposed of with the cultureware.The probe is disposed of with the used cultureware, no time is spentrecovering the probe for cleaning, revalidating and reuse.

In operation the probe 26, for example, a solid gel filled electrode, ismounted in a holder 28 (FIG. 23) through which the media to be sensedflow. The electrode in the holder is fluidically connected to thecultureware circuit, mounted in the cultureware module, the circuit ischecked for fluidic integrity, and sterilized with the completedcultureware (ethylene oxide, EtO). After sterilization, QC checks areperformed on the EtO process to provide high confidence ofsterilization. When an operator wishes to culture cells, the culturewareis removed from the pouch, loaded on the instrument and fluid isintroduced into the cultureware. A period of time is given to re-hydratethe electrode. The cultureware is brought to operating conditions, theelectrode is calibrated and then used to control pH in the cultureware.When the cell culture is complete, the operator disposes of thecultureware and the probe. Although the probe has been described as asolid gel electrode other probe types could be used (e.g. an ISFET,liquid filled, immobilized phenol matrix, fluorescence, etc).

Referring to the flow diagram of FIG. 23, pump 16 moves fresh basalmedia into the cultureware at media line 210. Media line 210 isconnected to a user provided container of fresh media to provide thegrowth nutrients to the cell culture that are pumped into thedisposable. Outflow line 214 is connected to a user provided containerto collect the waste or spent media being pumped out of the disposable.Factor line 212 is connected to a user provided container of growthfactors that are pumped into the disposable. EC inoculate can be addedat 220 and IC sample at 216. Product harvest is removed at 126. Thecells are harvested at 218. Harvest line 218 is a pre-attached containerthat is part of the disposable that is used to collect the product thatis pumped out of the disposable. Pump 16 has multiple lines 210, 214,212 and 126. Because the pump of the present invention has a commonfixed axial shaft and individual servo driven rotors, the control of theflow of each can be independent, allowing one channel or flow to beincreased while another decreased.

As shown in FIG. 25, a bioreactor 170 having a flexible outer body 172allows for physical movement of the cell growth substratum (hollowfibers, membrane or other suitable matrix) when a resultant torqueing orbending moment is applied to the bioreactor ends. Flexible outer body172 allows for the bioreactor case to be flexed causing fiber movement.This fiber movement enhances the release of cells that have attached tothe side of the bioreactor matrix. The cells can then be harvested byflushing either after or during the manipulation. This method canprovide increased efficiency of cell harvest at high cell viabilitieswithout the use of chemical or enzymatic release additives.

A bioreactor can be constructed using an outer housing that incorporatesa flexible center section. This center section consists of a flexible,non-permeable tubing that allows each end of the bioreactor to bemanipulated thus causing movement of the growth matrix. The purpose ofthis movement is to release the attachment or clumping of cell productson the extra-capillary (EC) side of the fibers. The cell products canthen be flushed from the EC via the access port at each end of thebioreactor.

Harvesting cells from a matrix-containing bioreactor such as a hollowfiber bioreactor has been difficult to accomplish. Typically cells aresticky and attach themselves to the fibers or to other cells and formclusters. Rapid flushing of media through the EC to hydraulically forcethe cells free and into the harvest stream is the most basic method ofharvesting cells from the EC space. Typically the quantity of cellsharvested is low because the flushing media tends to shunt through theEC and flush cells only from the limited fluid path.

Another method is to physically shake or impact the outer housing torelease the cells or clumps of cells. This practice may cause physicaldamage to the bioreactor or its associated components. Another methodincludes the use of chemicals to disrupt the adhesion of cells to thefibers or to disrupt the clumps of cells. Adding chemicals to acontrolled process may cause adverse effects on cell viability and canintroduce an unwanted agent in the down-stream processing.

Referring to FIGS. 26-31, various views of touch display screenillustrate the different interactive steps during control process of thesystem of the present invention. FIG. 26 shows a system overview screenwhich highlights current conditions. FIG. 27 illustrates a run sequencescreen which directs the operator through the culture process. FIG. 28illustrates log data which the operator can review and which isavailable to build the batch record. FIG. 29 shows a method forinputting alpha-numeric data. FIGS. 30 and 31 show operator interactionscreens to assist in operations (factor addition and pH probecalibration). On line help screens aid the operator for correctoperation.

Some examples for which the system of the present invention can be usedare:

-   -   The production of monoclonal antibodies from hybridoma cell        lines.    -   The expansion of autologous patient-derived blood cells        including immune cells for therapeutic application.    -   The expansion of patient derived somatic cells for subsequent        re-infusion back into patients for therapeutic purposes. A        specific example already available for therapeutic application        in patients is the harvesting and expansion of patient specific        cartilage cells (chondrocytes) followed by re-infusion of those        cells back into a region containing damaged articular cartilage.    -   The expansion of patient derived or generic multipotent cells,        including embryonic stem cells, adult stem cells, hematopoeitic        stem or progenitor cells, multi or pleuripotent cells derived        from cord blood for therapeutic purposes.    -   The expansion of somatic or germline cells as in applications 2,        3 and 4 in which the cells have been genetically modified to        express novel cellular components or to confer on them other        beneficial properties such as novel receptors, altered growth        characteristics or genetic features, followed by introduction of        the cells into a patient for therapeutic benefit. An example is        the expansion of patient specific fibroblasts genetically        modified to express growth factors, clotting factors, or other        biologically active agents to correct inherited or acquired        deficiencies of such factors.

At present, the system of the present invention fully integrates theconcept of disposable cultureware into automated process control formaintaining and expanding specialized (autologous or other) cell linesfor a duration of any time needed. To accomplish this, the system of thepresent invention was designed for EC space fluid flow that enhancescell growth in high density perfusion culture, yet remains completelyclosed and disposable. The integrated pre-assembled cultureware, whichconsists of all tubing, bioreactor, oxygenator, pH probe, is enclosed ina single unit that easily snaps into the apparatus. In addition to thiserror-proof, quick-load design, the entire cultureware unit enclosed bythe casing becomes the cell culture incubator with temperature controlregulated through automated process control of the instrument. Pumps andfluid control valves facilitate disposability and error-proofinstallation, eliminating the possibility of technician mistakes.Finally, during the course of any culture, the closed system hasrestricted access except for trained and authorized personnel.Manipulations or sampling, outside of program parameters, requirepassword and bar code access before they can be implemented.

Each unique cell line must be cultured, cell secretions harvested andpurified separately. In order to manage a large number of unique celllines, as for example might be required for the production of largenumbers of autologous cell therapeutic products or large numbers ofunique monoclonal antibodies, a considerable number of instruments wouldbe needed. Compactness of the design and the amount of ancillary supportresources needed become an important facilities issue. Small stirredtank systems require a means of steam generation and distribution (forsteam-in-place sterilization) or autoclaves to sterilize the vessels andsupporting plumbing. To support a large number of units becomes alogistics problem for the facility. The system of the present inventionhas no such requirement. Larger scale cell culture is historically donein segregated steps that often require separate types of equipment.Manual handling, storage and tracking is needed for all these steps asthe culture expands and product is harvested. The method of the presentinvention integrates these steps into a continuous, fully integratedsequential process. This eliminates the handling risk and facilitatesthe data gathering required for thorough documentation of the entireprocess.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. It ispreferred therefore, that the present invention be limited not by thespecific disclosure herein, but only by the appended claims.

1. A cell culture system for the production of cells and cell derivedproducts comprising: a reusable instrumentation base deviceincorporating hardware to support cell culture growth; and at least onedisposable cell cultureware module removably attachable to saidinstrumentation base device, said module including a cell growthchamber.
 2. The cell culture system of claim 1, wherein saidinstrumentation device includes a pump for circulating cell culturemedium through the at least one cultureware module.
 3. The cell culturesystem of claim 2, wherein the pump moves growth factor or othersupplements into the cell growth chamber and removes product harvestfrom the cell growth chamber.
 4. The cell culture system of claim 2,wherein said instrumentation device includes a plurality of rotaryselection valves to control the medium flow through the at least onecultureware module.
 5. The cell culture system of claim 1, wherein saidinstrumentation device includes a cool storage area for storing growthfactor or other supplements and product harvest, and wherein saidinstrumentation device includes a heating mechanism for heating the cellgrowth chamber to promote growth and production.
 6. The cell culturesystem of claim 5, wherein said at least one cultureware module includesan inlet and outlet port, said inlet and outlet ports being constructedand arranged to align with air ports of said instrument device such thatthe heat exchange mechanism forces heated air into said at least onecultureware module from said instrument device.
 7. The cell culturesystem of claim 1, wherein said instrumentation base device includes amulti-channel peristaltic pump for moving fresh basal media into said atleast one cultureware module, wherein said multi-channel peristalticpump has discrete, variable output control for each channel, and whereinsaid at least one cultureware module further comprises a pump cassette,wherein said pump cassette is structured to hold multiple peristaltictubing segments, and wherein said pump cassette is insertable into aninterface on said instrumentation base device and allows for said tubingsegments to be loaded into said multi-channel peristaltic pump in saidinstrumentation base device at the same time.
 8. The cell culture systemof claim 2, wherein said at least one cultureware module includes a gasblending mechanism in communication with the cell growth chamber,wherein the cell culture system further comprises a pH sensor disposedin said at least one cultureware module to control the pH of the cellculture medium, wherein the gas blending mechanism includes a gasexchange cartridge that provides oxygen and adds or removes carbondioxide to the medium to support cell metabolism, wherein the gasexchange cartridge has an inlet end and a discharge end, and wherein thecell culture system further comprises a carbon dioxide sensor in fluidcommunication with the discharge end of the gas exchange cartridge formeasuring the carbon dioxide level of the cell culture medium.
 9. Thecell culture system of claim 1, wherein said at least one culturewaremodule is pre-sterilized.
 10. The cell culture system of claim 1,wherein said at least one cultureware module includes a plurality ofinterface features integrated into the module that mate with instrumentinterface features in said instrumentation device.
 11. The cell culturesystem of claim 2, wherein said at least one cultureware module includessensors for sensing fluid circulation rate, temperature and pH of thecell culture medium.
 12. The cell culture system of claim 1, wherein thecell growth chamber comprises a bioreactor that provides cell space andmedium component exchange, and wherein the bioreactor has a flexibleouter body.
 13. The cell culture system of claim 1, wherein said atleast one cultureware module includes a fluid cycling unit disposedtherein to cycle and maintain fluid volumes within the cell growthchamber, and wherein the fluid cycling unit includes a non-rigidreservoir and a second flexible reservoir in fluid communication withthe first reservoir to cause elevated pressure in the first reservoir.14. The cell culture system of claim 1, further comprising a pluralityof disposable containers for harvest collection and flushing removablyconnected to said at least one cultureware module.
 15. A method for theproduction of cells and cell products in a highly controlled,contaminant-free environment comprising the steps of: providing at leastone disposable cultureware module, said module including a cell growthchamber; providing a reusable instrumentation base device incorporatinghardware to support cell culture growth, said base device including amicroprocessor control and a pump for circulating cell culture mediumthrough the cell growth chamber; removably attaching said at least onecultureware module to said instrumentation base device; introducingcells into the cell growth chamber; fluidly attaching a source of cellculture medium to said at least one cultureware module; programmingoperating parameters into the microprocessor control; operating the pumpto circulate the cell culture medium through the cell growth chamber togrow cells or cell products therein; harvesting the grown cells or cellproducts from the cell growth chamber; and disposing of said at leastone cultureware module.
 16. The method of claim 15, wherein said atleast one cultureware module includes a pH sensor disposed therein andfurther comprising the step of controlling the pH of the cell culturemedium, wherein the method further comprises the step of regulating thecell culture medium feed rate control of the medium, wherein the step ofregulating the cell culture medium feed rate control includes monitoringcarbon dioxide levels in the cell growth chamber to calculate lactateconcentration of the cell culture medium, and wherein the step ofregulating includes calculating an initial bicarbonate level of the cellculture medium and utilizing the measured pH and carbon dioxide level ofthe cell culture medium to calculate the lactate concentration.
 17. Themethod of claim 15, further comprising the step of pumping highmolecular weight factor into the cell growth chamber, and wherein saidinstrumentation base device includes a cool storage area and furthercomprising the step of storing the high molecular weight factor andproduct harvest in the cool storage area.
 18. The method of claim 15,wherein said at least one cultureware module includes a fluid cyclingunit disposed therein and further comprising the step of cycling andmixing fluid of the cell culture medium within the cell growth chamber,wherein cycling is achieved by utilizing a sealed flexible reservoir forthe extracapillary (EC) reservoir and the step of cycling comprisescycling the cell culture medium in and out of the flexible reservoir,and wherein the step of cycling further comprises using a secondflexible reservoir to apply indirect pressure to the EC reservoir toeffect cycling of the cell culture medium.
 19. A method for theexpansion or growth of cells, comprising introducing cells into the cellgrowth chamber of the cell culture system of claim 1, growing cells orcell products from the introduced cells, and harvesting the grown cellsor cell products from the cell growth chamber.
 20. The method of claim19, further comprising infusing or implanting the grown cells into ahuman or animal.